糖尿病大鼠视网膜中缺氧诱导因子-1α的表达与超微结构研究

目的 研究糖尿病大鼠视网膜中缺氧诱导因子-1α（HIF-1α）的表达与超微结构变化。方法 将132只SD大鼠随机分为对照组与糖尿病视网膜病变（DR）组，DR组采用链脲佐菌素（STZ）一次性腹腔注射建立糖尿病模型。应用免疫组织化学SP法和RT-PCR技术分别于1、3、6个月检测视网膜中HIF-1α蛋白及其mRNA的表达，透射电镜观察视网膜的超微结构。结果 免疫组织化学与RT-PCR检测结果均显示，HIF-1α蛋白及其mRNA在对照组视网膜中不表达，在DR组中呈进行性表达增强（P〈0．05）；透射电镜观察：正常对照组大鼠视网膜血管未见异常，DR组随着病程发展逐渐出现视网膜血管内皮细胞肿胀，基底膜厚薄不均。结论 DR中HIF-1α的异常表达和超微结构改变与视网膜缺氧关系密切，两者与DR的发生发展密切相关。     

缺氧诱导因子-1α在早期糖尿病大鼠视网膜神经细胞中的表达

目的研究缺氧诱导因子-1α(hypoxiainduciblefactor-1α,HIF-1α)在早期糖尿病大鼠视网膜中的表达,以探讨其在糖尿病性视网膜神经细胞病变发生及发展中的作用。方法用链脲佐菌素制作糖尿病大鼠模型,在制模成功后1周、2周、4周、6周、8周、10周及12周取眼球组织,以年龄匹配正常大鼠作为对照组,分别以免疫组织化学染色及Westernblotting方法检测视网膜组织中HIF-1α表达的位置及表达量;同时,以Real-timeRT-PCR方法检测视网膜组织中血管内皮细胞生长因子(vascularendothelialgrowthfac-tor,VEGF)的mRNA表达水平。结果糖尿病模型诱导成功后1周,视网膜组织即有HIF-1α表达,主要位于节细胞层及内核层的细胞核内,4周时阳性细胞数大于1周(12.34±0.41vs9.32±0.74),6~10周达到高峰(分别为16.51±0.35,45.33±0.52和37.31±0.43),12周下降(9.82±0.54)。Westernblotting显示HIF-1α蛋白表达模式与免疫组织化学相似,1周时即有表达,6~10周达到高峰,12周下降。Real-timeRT-PCR结果显示视网膜组织VEGF的mRNA水平在基础状态下少量表达,糖尿病诱导成功后表达水平逐渐提高,8周达到高峰,之后开始下降。结论HIF-1α及其下游基因VEGF在早期糖尿病视网膜神经细胞中的瞬时高表达而不是持续表达,可能是早期糖尿病视网膜神经细胞损害的原因。     

糖尿病大鼠视网膜中缺氧诱导因子-1α和血管内皮生长因子表达的研究

目的:检测缺氧诱导因子-1α(HIF-1α)与血管内皮生长因子(VEGF)在糖尿病大鼠视网膜中的表达,探讨其在糖尿病视网膜病变(DR)发生发展中的作用。方法:SD大鼠120只随机分为对照组与DR组,DR组采用链脲佐菌素(STZ)一次性建立糖尿病模型。应用免疫组化SP法和RT-PCR技术分别于1,3,6mo检测视网膜中HIF-1α与VEGF蛋白及其mRNA的表达变化。结果:HIF-1α与VEGF蛋白及其mRNA在对照组视网膜中不表达,在DR组中呈进行性表达增强(P<0.05);DR组中HIF-1α与VEGF的表达呈正相关(r=0.605,P<0.05)。结论:DR视网膜中HIF-1α与VEGF表达增强,与DR的发生发展密切相关。     

缺氧诱导因子-1α小干扰RNA对糖尿病大鼠视网膜血管内皮生长因子mRNA抑制作用的动态观察

目的 观察缺氧诱导因子-1α(HIF-1α)特异性小干扰RNA(siRNA)对糖尿病大鼠视网膜组织中血管内皮生长因子(VEGF)mRNA表达的抑制作用.方法 随机对照研究.以pSilencer 2.1-U6neo为质粒载体,构建HIF-1α siRNA重组质粒.雄性Sprague-Dawley大鼠54只,随机分为正常对照组和实验组,分别为15、39只大鼠.实验组大鼠尾静脉注射链尿佐菌素(STZ)诱导建立糖尿病动物模型,再分为糖尿病模型组、空载体组和基因治疗组,分别为15、12、12只大鼠.脂质体Lipofectamine TM 2000介导,空载体组和基因治疗组分别转染pSilencer空载体质粒和HIF-1αsiRNA重组质粒,糖尿病模型组和正常组对照组不做转染.采用实时逆转录(RT)-聚合酶链式反应(PCR)检测各组大鼠VEGF mRNA的表达.分别于干扰后24、48、72 h,1周时计算VEGF mRNA的抑制效率.采用单因素方差分析和LSD-t检验进行统计学分析.结果 HIF-1α siRNA重组质粒经酶切、测序鉴定,确定为目的 序列.实时RT-PCR检测结果显示,正常对照组仅见弱的VEGF mRNA表达,糖尿病模型组及空载体组表达明显上调,基因治疗组表达下调;各时间点空载体组与糖尿病模型组比较,差异无统计学意义(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980),基因治疗组较糖尿病模型组及空载体组表达下调,差异有统计学意义(t=8.768、13.695、11.285、8.253,t=9.437、13.554、11.436、8.228;P值均=0.000);VEGF mRNA抑制率24、48、72 h和1周时分别为32.76%、43.60%、47.70%、50.86%.结论 HIF-1α siRNA重组质粒能有效抑制糖尿病大鼠视网膜中VEGFmRNA的表达.

Abstract：

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

缺氧诱导因子1α对早期糖尿病视网膜病变状态下人粒细胞系白血病细胞表面黏附分子CD18表达的调节

目的　观察在早期糖尿病视网膜病变(DR)状态下,缺氧诱导因子1α（HIF-1α）对细胞表面黏附分子CD18表达以及白细胞和血管内皮细胞黏附的影响。方法　收集早期DR患者及年龄匹配的健康人外周血血清,用于体外培养人粒细胞系白血病细胞（HL60）和恒河猴视网膜血管内皮细胞系细胞（RF/6A）。分为糖尿病血清培养组（B组）、糖尿病＋HIF 1α反义寡核苷酸组（ASODN）（C组）、糖尿病＋HIF 1α 正义寡核苷酸组（SODN）（D组）,健康人血清培养细胞为正常对照组（A组）。以流式细胞仪和Real-time PCR分别检测HL60细胞表面CD18蛋白阳性细胞比例和CD18 mRNA的表达水平,以虎红染色法观察HL60细胞和RF/6A细胞的黏附率。结果　A、B、C、D组CD18阳性细胞比例分别为17.06±6.01、42.23±2.60、25.33±3.05、32.40±10.57,各组之间差异有统计学意义（F=36.47,P＜0.001）。和A组比较,B、C、D组CD18 mRNA水平分别是A组的21.05±2.07、2.23±0.96、25.07±2.27倍,各组之间差异有统计学意义（F＝180.34,P＜0.001）。A、B、C、D组细胞黏附率分别为0.06±0.002、0.09±0.10、0.05±0.007、0.07±0.01,各组之间差异有统计学意义（F=13.06,P=0.002）。结论　在体外培养条件下,糖尿病血清可以促进HL60细胞表达CD18蛋白和CD18mRNA,促进HL60细胞和血管内皮细胞的黏附,HIF-1α表达被抑制后,这种促进作用减弱。HIF-1α对糖尿病状态下细胞表面黏附分子CD18表达及白细胞和血管内皮细胞之间的黏附具有调节作用。      

缺氧诱导因子1α特异性小干扰RNA对髓样细胞表面黏附分子CD18及神经损伤诱导蛋白-1表达的影响

目的 观察早期糖尿病视网膜病变（DR）状态下,以pSUPER为载体的缺氧诱导因子1α（HIF1α）特异性小干扰（siHIF1α）RNA对髓样细胞表面黏附分子CD18及神经损伤诱导蛋白-1（Ninj-1）表达的影响。方法 实验分为健康人血清培养组（A组）、糖尿病血清培养组（B组）、糖尿病血清培养联合pSUPERH1 siHIF1α转染组（C组）及糖尿病血清培养联合pSUPER空载体组（D组）进行。A组采用健康志愿者血清培养人髓样细胞系白血病细胞系（K562）细胞;B、C、D组均采用DR患者血清培养K562细胞。C、D组在加入DR患者血清前24 h分别转染pSUPERH1 siHIF1α及pSUPER空载体。采用流式细胞仪检测各组K562细胞表面的CD18、Ninj-1表达。行细胞黏附实验,检测各组K562细胞与恒河猴视网膜脉络膜血管内皮细胞系（RF/6A）细胞黏附率。结果 A、B、C、D组K562细胞表面CD18表达比较,4组间差异有统计学意义（F=14.33,P=0.01）。组间CD18表达两两比较,B组明显高于A组（P=0.001）;C组明显低于B组（P=0.001）和D组（P=0.02）;C组与A组间无明显差异（95%可信区间=-14.89~2.13,P=0.12）。A、B、C、D组K562细胞表面Ninj-1表达比较,4组间差异有统计学意义（F=39.38,P=0.001）。组间Ninj-1表达两两比较,B组明显高于A组（P=0.00）;C组与B组间无明显差异（P=0.06）;C组与D组间也无明显差异（P=0.49）。A、B、C、D组K562细胞与RF/6A细胞黏附率比较,4组间差异有统计学意义（F=20.62,P=0.00）。组间K562细胞与RF/6A细胞黏附率两两比较,B组明显高于A组（P=0.00）;C组较B组明显下降（P=0.01）,较A组明显提高（P=0.002）;B组与D组间无明显差异（P=0.68）。结论 早期DR状态下,以pSUPER为载体的siHIF1α RNA可降低K562细胞表面CD18的表达,但对Ninj-1表达无明显影响。     

缺氧诱导因子-1α、血管内皮生长因子在视网膜母细胞瘤中的表达及意义

肿瘤的生长和转移依赖肿瘤细胞对缺氧的适应和新生血管的形成,而肿瘤新生血管的形成受多种因子的调控.缺气门诱 导因子-1α（HFIF-1α)是缺氧条件下肿瘤细胞生长的一种转录因子.     

低氧诱导因子-1α在胚胎及出生后大鼠视网膜中的表达变化

目的 观察大鼠视网膜胚胎期及出生后早期发育过程中低氧诱导因子-1α(HIF -1α)的时空表达,探讨其表达变化与视网膜发育的关系。方法 实验大鼠分为孕12、16、20 d 组,出生后1、5、10 d组及成年组,每组各5只,共35只大鼠。用免疫组织化学方法、半定量逆转录聚合酶联反应（RT-PCR）方法检测视网膜H IF-1α蛋白及HIF-1αmRNA的表达。结果 胚胎期大鼠视网膜神经上皮层及视网膜色素上皮层均有HIF 1α阳性表达,出生后发育早期视网膜神经上皮层及视网膜色素上皮层也可见HIF-1α阳性表达,以神经节细胞、内网层更明显。随着发育进展,其阳性表达主要位于视网膜节细胞层。大鼠视网膜HIF-1α蛋白及mRNA的表达在胚胎期最高、出生后发育期逐渐下降、成年期最低,其差异有统计学意义（P<0.01）。结论 大鼠视网膜胚胎及出生后发育过程中HIF-1α的表达存在时空上的变化,这种变化可能与大鼠视网膜胚胎期及出生后早期的发育密切相关。     

缺氧诱导因子-1α在早期糖尿病大鼠角膜上皮细胞中的表达

目的:观察缺氧诱导因子-1α(HIF-1α)在早期糖尿病大鼠角膜上皮细胞中的表达以探讨其在糖尿病性角膜上皮细胞病变发生及发展中的作用。方法:用链脲佐菌素(STZ)制作糖尿病大鼠模型,在制模成功后1,2,4,6,8,10及12wk取眼球组织,以年龄匹配正常大鼠作为对照组,分别以免疫组织化学染色及Westernblotting方法检测角膜上皮细胞中HIF-1α表达的位置及表达量。结果:糖尿病模型诱导成功后1wk,角膜上皮细胞中即有HIF-1α表达,主要位于基底细胞的细胞核内,4wk时阳性细胞数大于1wk(21.80±1.93vs15.30±2.05),6～10wk达到高峰(6,8和10wk分别为49.00±2.82,90.80±4.23,51.40±5.21),12wk下降(14.40±2.06)。Westernblotting分析显示HIF-1α蛋白表达模式与免疫组织化学相似,1wk时即有表达,6～10wk达到高峰,12wk下降。结论:HIF-1α在早期糖尿病角膜上皮细胞中的瞬时高表达而不是持续表达,可能是早期糖尿病角膜上皮细胞病变的原因。     

缺氧诱导因子1α在慢性高眼压下大鼠视网膜中的表达

目的了解缺氧诱导因子1((hypoxia-inducible factor-1α,HIF-1α)在实验性慢性高眼压大鼠视网膜中的表达并探讨其在青光眼视网膜损伤中的作用。方法60只大鼠随机分为假手术对照组;高眼压3d组;高眼压7d组;高眼压14d组;高眼压21d组;高眼压28d组。浅层巩膜静脉烧灼法制成慢性高眼压模型,通过逆转录聚合酶链式反应(RT-PCR)方法和蛋白印迹法(Western-blot)研究HIF-1在慢性高眼压下不同时段视网膜中的表达情况。结果HIF-1αmRNA和蛋白在高眼压的视网膜表达较正常视网膜明显提高(P<0.05)。高眼压后7天达到高峰,可维持28天。结论HIF-1参与了慢性高眼压的视网膜的损伤的信号转导过程,为青光眼视神经损伤的缺氧学说提供了直接证据。     

缺氧诱导因子对糖尿病视网膜病变调控作用的研究现状与进展

以血管内皮生长因子（VEGF）为靶点的干预治疗已成为目前治疗糖尿病视网膜病变（DR）的特异性强且有效的方法。但部分患者经抗VEGF药物治疗后无应答或应答不良,并且其消除水肿和改善视力的作用在同一患者中的表现似乎也不稳定。缺氧诱导因子-1α （HIF-1α）作为VEGF重要的上游转录调控因子,是组织低氧状态下表达的具有氧...     

缺氧时视网膜血管内皮细胞缺氧诱导因子-1α及其mRNA的时序性表达

目的探讨在缺氧状态下视网膜血管内皮细胞缺氧诱导因子-1α(HIF-1α)及其mRNA表达的时空规律.方法对分离的牛视网膜血管内皮细胞分别进行常规和CoCl2模拟缺氧培养,采用免疫组化和逆转录多聚酶链反应(RT-PCR)检测不同缺氧时间HIF-1α及其mRNA的表达.结果正常对照组HIF-1α有极低表达,在缺氧1h时表达量显著上升,4h达到高峰,16h后下降.与对照组比较,各缺氧组HIF-1α的表达均有显著性差异(P＜0.01).但各缺氧组HIF-1αmRNA的表达无明显差异.结论视网膜血管内皮细胞在缺氧早期HIF-1 α表达有短暂、迅速的增加,但其mRNA的表达无明显变化,提示缺氧可以促进视网膜血管内皮细胞HIF-1α的表达,但不是在基因的转录水平.     

臭氧对STZ诱导的糖尿病大鼠视网膜表达VEGF, HIF-1α与PEDF的影响

目的：通过测定经臭氧灌肠治疗后的糖尿病大鼠视网膜及血清中血管内皮生长因子（ VEGF ）、缺氧诱导因子－1α（ HIF－1α）与色素上皮衍生因子（ PEDF）的表达差异,了解臭氧在早期糖尿病视网膜病变治疗的作用。方法：选取70只雄性SD大鼠中的10只做为正常对照组,予以正常饮食;余大鼠经适应性喂养后通过腹腔一次性注射链脲佐菌素（50mg／ml）制作糖尿病模型。将造模成功的大鼠模型随机分成模型对照组（B组）、氧气治疗组（C组）及臭氧治疗组（D组）,其中臭氧治疗组给予臭氧灌肠治疗（2次／wk,共计1mo）,氧气治疗组给予同等剂量及频次的氧气进行灌肠治疗,治疗1mo 后取视网膜及血清,使用免疫组化法检测视网膜中 VEGF 的表达,使用ELISA及RT－PCR的方法检测视网膜及血清中 VEGF、HIF－1α与PEDF的含量。结果：免疫组化结果显示VEGF主要在内层视网膜表达,而臭氧治疗组VEGF表达接近于空白对照组;血清与视网膜中VEGFmRNA 表达在四组间均有统计学差异（F ＝23．923;P＝0．000）,其中臭氧治疗组VEGF的表达接近于空白对照组,但差异仍有统计学意义（P＜0．05）;而模型对照组同氧气治疗组的 VEGF 表达亦无统计学差异（ P＞0．05）;相对于空白对照组,臭氧治疗组HIF－1α表达下降（P＜0．05）,而在模型对照组与氧气治疗组间无统计学差异（ P＞0．05）。 PEDFmRNA在四组间的表达均有差异,但差异均无统计学意义（P＞0．05）。结论：臭氧灌肠治疗可以有效的降低血清及视网膜中VEGF和HIF－1α的表达,可以一定程度的抑制早期糖尿病视网膜病变的发展。     

缺氧诱导因子-2α在增生型糖尿病视网膜病变新生血管形成中的作用

目的观察HIF-2α在增生型糖尿病视网膜病变(PDR)纤维血管膜中的表达,初步探讨HIF-2α在PDR新生血管形成中的作用。方法回顾性临床研究。2014年7月至2015年7月于青岛大学附属医院眼科行玻璃体切割手术的PDR患者57例60只眼纳入研究。根据手术前是否行玻璃体腔注射抗VEGF药物治疗将患眼分为PDR未注药组和PDR注药组,分别为30例32只眼、27例28只眼。PDR注药组患眼手术前2~7 d玻璃体腔注射10 mg/ml雷珠单抗0.05 ml(含雷珠单抗0.5 mg)。选取同期特发性黄斑前膜患者18例18只眼作为对照组。于玻璃体切割手术中获得PDR纤维血管膜、黄斑前膜病理标本。免疫组织化学染色法及荧光定量PCR(RT-PCR)检测各组病理标本中HIF-2α、Delta样配体4(Dll4)、VEGF的表达。多组间相关因子表达差异比较采用Kruskal-Wallis检验。两变量间关系行Spearman相关性分析。结果免疫组织化学染色结果显示,PDR未注药组、PDR注药组纤维血管膜中HIF-2α、Dll4、VEGF蛋白表达均呈阳性。PDR未注药组HIF-2α、Dll4、VEGF蛋白相对表达量均高于PDR注药组,差异均有统计学意义(t=4.36、6.01、4.82,P=0.000、0.008、0.016)。RT-PCR检测结果显示,PDR未注药组、PDR注药组、对照组纤维血管膜、黄斑前膜中HIF-2α、Dll4、VEGF mRNA相对表达量比较,差异均有统计学意义(H=18.81、19.60、20.50,P=0.000、0.000、0.000)。其中,PDR未注药组、PDR注药组HIF-2α、Dll4、VEGF mRNA相对表达量均显著高于对照组;与PDR未注药组比较,PDR注药组HIF-2α、Dll4、VEGF mRNA相对表达量均降低。Spearman相关性分析结果显示,HIF-2α与Dll4、VEGF mRNA相对表达量呈显著正相关(r=0.95、0.87,P<0.05)。结论PDR患眼纤维血管膜中HIF-2α表达高于对照组,且与DLL4、VEGF的表达均呈显著正相关。     

糖尿病视网膜病变患者外周血中性粒细胞表面CD18的表达

目的研究不同程度糖尿病视网膜病变（DR）患者外周血中性粒细胞表面CD18的表达。方法38例DR患者,其中包括1期DR组10例,2～4期DR组14例,5期DR组14例。正常对照组10例。流式细胞仪测量受试者外周血中性粒细胞表面CD18的表达。结果4个组CD18平均荧光强度（MCF）之间的差异有统计学意义（P〈0．01）。CD18的表达水平最高为5期DR组,其次为2-4期DR组及1期DR组,最低为正常对照组（趋势检验,P〈0．01）。调整混杂因素后这种趋势仍然存在。多元线性回归分析显示CD18的MCF水平与DR分期存在显著相关性（β＝0．33,P=0．01）。结论外周血中性粒细胞表面CD18水平的提高可能是2型糖尿病患者DR进展的临床标志。     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

目的 观察色素上皮衍生因子(PEDF)对糖尿病大鼠视网膜谷氨酸代谢的影响.方法 Sprague_Dawley大鼠78只,分为模型组、模型对照组、PEDF干预组(干预组)、干预对照组,实验结束时去除血糖恢复鼠和实验期间死亡鼠,每组以12只大鼠作为统计样本.模型组、干预组、干预对照组大鼠采用链脲佐菌素诱导糖尿病大鼠模型.模型组大鼠不作任何干预,模型对照组为相同月龄的正常大鼠.干预组大鼠左眼玻璃体腔注射0.1 μg/μl的PEDF 5.0μl,干预对照组大鼠左眼玻璃体腔注射相同容积的磷酸盐缓冲液.采用蛋白免疫印迹法(Western bolt)和实时荧光聚合酶链式反应(PCR)法检测视网膜L-谷氨酸-L-门冬氨酸转运体(GLAST)表达的变化,高压液相色谱法(HPLC)观察视网膜谷氨酸的含量变化.将体外培养的大鼠视网膜Müller细胞随机分为对照组、实验组、PEDF干预组(干预组)和干预对照组,荧光免疫法和实时荧光PCR法检测Müller细胞GLAST表达的改变,根据[3H]标记的D,L-谷氨酸摄入量判断Müller细胞的摄取功能.结果 实时荧光PCR法和Western bolt检测结果显示,相对于模型对照组大鼠,模型组大鼠视网膜GLAST表达降低(实时荧光PCR法:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05),视网膜谷氦酸含量升高(t=4.01,P＜0.05);干预组大鼠视网膜GLAST表达与干预对照组视网膜GLAST表达比较,干预组大鼠视网膜GLAST的表达升高(实时荧光PCR法:t=3.56,P＜0.05;Western blot:t=3.52,P＜O.05);视网膜谷氨酸含量下调(t=4.36,P＜0.05).实时荧光PCR法和荧光免疫法检测结果显示,高糖可以降低视网膜Müller细胞GLAST的表达(实时荧光PCR法:t=3.48,P＜O.05;荧光免疫法:t=4.72,P＜O.05);[3H]标记的D,L-谷氨酸摄人量结果显示,高糖可以下调视网膜Mü1ler细胞GIAST的功能(t=3.81,P＜0.05);经PEDF处理后,可以明显改善高糖状态下视网膜Müller细胞GLAST的表达(实时荧光PCR法:t=6.82,P＜O.01;荧光免疫法:t=3.72,P＜0.05)和对谷氨酸的摄取功能(t=4.14,P＜0.05).结论 PEDF可通过改善糖尿病大鼠视网膜Müller细胞中GLAST功能从而改善谷氨酸循环,抑制神经节细胞的死亡.

Abstract：

Objective To observe the effect of pigment epithelium-derived factor(PEDF)on glutamate metabolism in diabetic rat retina.Methods 78 Sprague-Dawley rats were randomly divided into the model group,model control group,PEDF intervention group and intervention control group.There were some dead and euglycemia rats at the end of experiment,so only 12 rats in each group were included in the statistical analysis.The diabetic retinopathy rat model of the model,PEDF intervention and intervention control group were induced with streptozotocin injection.The rats in the model group were not intervened.The monthly-age matched normal rats of model group were in the model control group.The left eyes of rats were received intravitreal injection with 5μl(0.1μg/μl)PEDF(PEDF intervention group)or 5μlphosphate buffer solution(intervention control group).The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography(HPLC).Cultured rat Mailer cells were divided into the control,experimental,PEDF intervention and intervention control group,GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques.The glutamate up-take activity of Müller cells was determined by intracellular[3H] labeled D,L-glutamate concentration with scintillation counting.Results Western blot and real-time RT-PCR showed that GLAST expression decreased(real-time RT-PCR:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05).glutamate content increased(t=4.01,P＜0.05)in model group compared with the model control group:GLAST expression increased(real-time RT-PCR:t=3.56,P＜0.05;Western blot:t=3.52,P＜0.05),glutamate content decreased(y=4.36,P＜0.05)in the PEDF intervention group compared with the intervention control group.Real-time RT-PCR and fluorescence immunofluorescenee showed that high glucose down-regulate GLAST expressions in Müller cells(real-time RT-PCR:t=3.48,P＜0.05;fluorescence immunofluoreseence:t=4.72,P＜0.05)and impair glutamate up-take activity of Müller cells(t=3.81,P＜0.05).Under high glucose conditions,PEDF up-regulated GLAST expression significantly(real-time RT-PCR:t=6.82,P＜0.01;fluorescence immunofluorescence:t=3.72,P＜0.05)and ameliorated the glutamate up-take activitv of Mailer cells(t=4.14,P＜0.05).Conclusions In diabetic rats,PEDF may improve the activitv of GLAST in Müller cells,thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

Objective To observe the effect of pigment epithelium-derived factor(PEDF)on glutamate metabolism in diabetic rat retina.Methods 78 Sprague-Dawley rats were randomly divided into the model group,model control group,PEDF intervention group and intervention control group.There were some dead and euglycemia rats at the end of experiment,so only 12 rats in each group were included in the statistical analysis.The diabetic retinopathy rat model of the model,PEDF intervention and intervention control group were induced with streptozotocin injection.The rats in the model group were not intervened.The monthly-age matched normal rats of model group were in the model control group.The left eyes of rats were received intravitreal injection with 5μl(0.1μg/μl)PEDF(PEDF intervention group)or 5μlphosphate buffer solution(intervention control group).The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography(HPLC).Cultured rat Mailer cells were divided into the control,experimental,PEDF intervention and intervention control group,GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques.The glutamate up-take activity of Müller cells was determined by intracellular[3H] labeled D,L-glutamate concentration with scintillation counting.Results Western blot and real-time RT-PCR showed that GLAST expression decreased(real-time RT-PCR:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05).glutamate content increased(t=4.01,P＜0.05)in model group compared with the model control group:GLAST expression increased(real-time RT-PCR:t=3.56,P＜0.05;Western blot:t=3.52,P＜0.05),glutamate content decreased(y=4.36,P＜0.05)in the PEDF intervention group compared with the intervention control group.Real-time RT-PCR and fluorescence immunofluorescenee showed that high glucose down-regulate GLAST expressions in Müller cells(real-time RT-PCR:t=3.48,P＜0.05;fluorescence immunofluoreseence:t=4.72,P＜0.05)and impair glutamate up-take activity of Müller cells(t=3.81,P＜0.05).Under high glucose conditions,PEDF up-regulated GLAST expression significantly(real-time RT-PCR:t=6.82,P＜0.01;fluorescence immunofluorescence:t=3.72,P＜0.05)and ameliorated the glutamate up-take activitv of Mailer cells(t=4.14,P＜0.05).Conclusions In diabetic rats,PEDF may improve the activitv of GLAST in Müller cells,thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.     

色素上皮衍生因子对糖尿病大鼠视网膜谷氨酸代谢的影响

Objective To observe the effect of pigment epithelium-derived factor(PEDF)on glutamate metabolism in diabetic rat retina.Methods 78 Sprague-Dawley rats were randomly divided into the model group,model control group,PEDF intervention group and intervention control group.There were some dead and euglycemia rats at the end of experiment,so only 12 rats in each group were included in the statistical analysis.The diabetic retinopathy rat model of the model,PEDF intervention and intervention control group were induced with streptozotocin injection.The rats in the model group were not intervened.The monthly-age matched normal rats of model group were in the model control group.The left eyes of rats were received intravitreal injection with 5μl(0.1μg/μl)PEDF(PEDF intervention group)or 5μlphosphate buffer solution(intervention control group).The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography(HPLC).Cultured rat Mailer cells were divided into the control,experimental,PEDF intervention and intervention control group,GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques.The glutamate up-take activity of Müller cells was determined by intracellular[3H] labeled D,L-glutamate concentration with scintillation counting.Results Western blot and real-time RT-PCR showed that GLAST expression decreased(real-time RT-PCR:t=8.86,P＜0.01;Western blot:t=3.42,P＜0.05).glutamate content increased(t=4.01,P＜0.05)in model group compared with the model control group:GLAST expression increased(real-time RT-PCR:t=3.56,P＜0.05;Western blot:t=3.52,P＜0.05),glutamate content decreased(y=4.36,P＜0.05)in the PEDF intervention group compared with the intervention control group.Real-time RT-PCR and fluorescence immunofluorescenee showed that high glucose down-regulate GLAST expressions in Müller cells(real-time RT-PCR:t=3.48,P＜0.05;fluorescence immunofluoreseence:t=4.72,P＜0.05)and impair glutamate up-take activity of Müller cells(t=3.81,P＜0.05).Under high glucose conditions,PEDF up-regulated GLAST expression significantly(real-time RT-PCR:t=6.82,P＜0.01;fluorescence immunofluorescence:t=3.72,P＜0.05)and ameliorated the glutamate up-take activitv of Mailer cells(t=4.14,P＜0.05).Conclusions In diabetic rats,PEDF may improve the activitv of GLAST in Müller cells,thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.     

缺氧诱导体外培养的人视网膜色素上皮细胞转录和表达缺氧诱导因子-1α的研究

目的探讨在不同缺氧时相人视网膜色素上皮(RPE)细胞转录和表达缺氧诱导因子-1α(HIF-1α)的情况。方法原代培养人RPE细胞,经鉴定后,选择第3—6代RPE细胞进行缺氧分析,分别在缺氧6、8、16、24、48h不同时相,利用半定量RT—PCR法及免疫荧光测定法检测人RPE细胞转录和表达HIF-1α的情况。结果半定量RT-PCR法和免疫荧光法检测结果均显示缺氧可以明显诱导入RPE细胞转录和表达HIF-1α。在缺氧8h时,HIF-1α转录和表达量达高峰,并可持续至24h后;缺氧48h时,人RPE细胞代谢产物增加,细胞形态发生改变,HIF-1α转录和表达量开始下降。结论缺氧可以明显诱导人RPE细胞转录和表达HIF-1α,这可能是发生脉络膜新生血管的重要始动因素。（中华眼科杂志,2005,41：312-316）